ARTICLE Received 10 Nov 2010 | Accepted 15 Feb 2011 | Published 15 Mar 2011 DOI: 10.1038/ncomms1239 Inflammation driven by tumour-specific Th1 cells protects against B-cell cancer Ole Audun Werner Haabeth1, Kristina Berg Lorvik1, Clara Hammarström2, Ian M. Donaldson3,4, Guttorm Haraldsen2, Bjarne Bogen1 & Alexandre Corthay1 The immune system can both promote and suppress cancer. Chronic inflammation and proinflammatory cytokines such as interleukin (IL)-1 and IL-6 are considered to be tumour promoting. In contrast, the exact nature of protective antitumour immunity remains obscure. Here, we quantify locally secreted cytokines during primary immune responses against myeloma and B-cell lymphoma in mice. Strikingly, successful cancer immunosurveillance mediated by tumour-specific CD4 + T cells is consistently associated with elevated local levels of both proinflammatory (IL-1α, IL-1β and IL-6) and T helper 1 (Th1)-associated cytokines (interferon-γ (IFN-γ), IL-2 and IL-12). Cancer eradication is achieved by a collaboration between tumour- specific Th1 cells and tumour-infiltrating, antigen-presenting macrophages. Th1 cells induce secretion of IL-1β and IL-6 by macrophages. Th1-derived IFN-γ is shown to render macrophages directly cytotoxic to cancer cells, and to induce macrophages to secrete the angiostatic chemokines CXCL9/MIG and CXCL10/IP-10. Thus, inflammation, when driven by tumour- specific Th1 cells, may prevent rather than promote cancer. 1 Centre for Immune Regulation, Institute of Immunology, University of Oslo and Oslo University Hospital Rikshospitalet, PO Box 4950 Nydalen, 0424 Oslo, Norway. 2 Department of Pathology, Institute of Pathology, Oslo University Hospital Rikshospitalet and University of Oslo, PO Box 4950 Nydalen, 0424 Oslo, Norway. 3 The Biotechnology Centre of Oslo, University of Oslo, PO Box 1125 Blindern, 0317 Oslo, Norway. 4 Department of Molecular Biosciences, University of Oslo, PO Box 1041 Blindern, 0316 Oslo, Norway. Correspondence and requests for materials should be addressed to A.C. (email: [email protected]). NATURE COMMUNICATIONS | 2:240 | DOI: 10.1038/ncomms1239 | www.nature.com/naturecommunications © 2011 Macmillan Publishers Limited. All rights reserved. ARTICLE NATURE COMMUNICATIONS | DOI: 10.1038/ncomms1239 he immune system can protect against cancer1. Elevated we provide evidence for a dual antitumour role of Th1-derived IFN-γ. numbers of intratumoral T cells predict long-term survival First, IFN-γ triggers tumouricidal activity of tumour-infiltrating for patients with advanced ovarian carcinoma and colorec- macrophages. Second, IFN-γ induces macrophages to secrete T 2,3 tal cancer . Yet, little is known about the exact nature of protec- the angiostatic chemokines CXCL9/MIG (monokine induced by tive antitumour immune responses. On the other hand, it is well IFN-γ) and CXCL10/IP-10 (IFN-γ inducible protein 10), which may established that chronic inflammation predisposes to cancer. halt tumour progression by inhibiting angiogenesis. Collectively, Proinflammatory cytokines such as interleukin (IL)-1 and IL-6 are our data suggest a cancer-protective role of inflammation driven by considered to be essential for tumour progression, and anti-inflam- tumour-specific Th1 cells. matory drugs have been suggested to treat cancer4–9. However, anti-inflammatory treatments may potentially suppress protective Results antitumour immunity. Strategies to fight malignancies should be The Matrigel cytokine assay. We have developed a method to based on stimulating rather than suppressing the ongoing immune quantify locally secreted cytokines during the early stages of an response against cancer. Therefore, it is crucial to better understand immune response against cancer in mice. The method is based on the interplay between immune cells, inflammation and cancer. s.c. inoculation of cancer cells embedded in a Matrigel collagen gel Tumour-specific CD4 + T cells orchestrate the immune response (Fig. 1a). Matrigel is derived from a murine sarcoma and therefore against cancer. CD4 + T cells are required for cytokine-mediated represents a genuine tumour cell microenvironment19. Matrigel, activation of tumour-specific cytotoxic CD8 + T cells, but they can which is liquid at + 4 °C, gelifies at body temperature and forms a also eliminate cancer in the absence of CD8 + T cells10,11. A recent gel plug containing the injected cancer cells18. The gel functions as study of patients with breast cancer concluded that high numbers of an extracellular matrix and contains the cytokines that are secreted CD4 + T cells in lymph nodes (LNs) predict disease-free survival12. locally by cancer cells and by infiltrating immune cells. At various time In lung and liver cancer, high CD4:CD8 T-cell ratios were associated points after injection, the Matrigel plug can be excised and dissolved with good prognosis13,14. However, CD4 + T cells may also suppress in vitro with collagenase. A cell-free supernatant containing secreted antitumour immunity15. To clarify the mechanism of cancer pre- cytokines that are present in the Matrigel extracellular matrix is vention by CD4 + T cells, we have used idiotype (Id)-specific T-cell obtained by centrifugation (Fig. 1a). Cytokines in the supernatant receptor transgenic (TCR-TG) mice, which were made homozygous are quantified by multiplex bead assays. An initial experiment to test for the severe combined immunodeficiency (SCID) mutation to the method revealed that numerous cytokines could be detected in prevent rearrangement of endogenous TCR chains11. In these mice, the Matrigel at day + 8, which represents the height of the antitumour tumour-specific CD4 + T cells recognize an Id peptide from the immune response18 (Supplementary Fig. S1). To identify cytokines variable region of the immunoglobulin light chain of the MOPC315 associated with successful cancer immunosurveillance, we designed myeloma, presented on major histocompatibility complex (MHC) a series of five experimental situations to compare cytokine levels class II molecules16. Id-specific TCR-TG SCID mice are resistant during successful versus failed immune responses against cancer. against subcutaneous (s.c.) inoculation with syngeneic MOPC315 myeloma cells or with Id-transfected F9 B-lymphoma cells, Tumour-specific CD4 + T cells control local cytokine levels. In whereas non-transgenic mice develop fatal tumours. Protection is the first experimental situation (myeloma ± specific T cells), we Id-specific, CD4 + T cell-mediated, and does not require the presence compared tumour-specific CD4 + T cells with CD4 + T cells that of B cells and CD8 + T cells11,17. recognized an irrelevant antigen, ovalbumin (OVA). Id-specific To study the mechanisms of cancer rejection by Id-specific TCR-TG SCID mice, OVA-specific TCR-TG SCID mice and SCID TCR-TG mice, we have developed a strategy consisting of embed- mice were injected s.c. with MOPC315 myeloma cells. Id-specific ding injected tumour cells in a collagen gel (Matrigel). The Matrigel TCR-TG SCID mice were protected against MOPC315 (Fig. 1b), as functions as an extracellular matrix in which infiltrating immune previously reported11. Control SCID mice, which lack T and B cells, cells can be analysed at various time points after injection. Using succumbed from cancer within 40 days. OVA-specific TCR-TG this method, we have reported the first characterization of a suc- SCID mice, which have T cells but cannot recognize Id, developed cessful primary antitumour immune response initiated by naïve tumours as quickly as SCID mice (Fig. 1b). For a cytokine assay, CD4 + T cells18. In brief, we could show that s.c. injected MOPC315 Id-specific and OVA-specific TCR-TG SCID mice were injected with myeloma cells were surrounded within 3 days by macrophages, MOPC315 in Matrigel (Fig. 1c–g). The total numbers of MOPC315 which captured tumour-specific antigens. Within 6 days, naïve cells and CD11b + macrophages in Matrigel at day + 8 were simi- Id-specific CD4 + T cells became activated in draining LN and sub- lar for both groups (Fig. 1d,e). However, when we used MHC class sequently migrated to the incipient tumour site. On recognition of II upregulation as an activation marker for macrophages18, acti- tumour-derived Id peptides presented on MHC class II molecules vated MHC class IIhigh macrophages were only found in Id-specific by macrophages, Id-specific CD4 + T cells were shown to secrete TCR-TG SCID mice (Fig. 1f). Quantification of 33 cytokines in interferon-γ (IFN-γ). Matrigel-infiltrating macrophages became acti- Matrigel revealed that the local concentration of 21 cytokines was vated by T cell-derived IFN-γ, and could kill MHC class II-nega- significantly higher in the presence of tumour-specific CD4 + T cells, tive myeloma cells directly18. However, in this previous report the that is, in tumour-resistant Id-specific TCR-TG SCID mice (Fig. 1g; exact function of IFN-γ was not fully defined and the involvement Supplementary Fig. S2). Notably, cytokine levels in serum were of other cytokines was not investigated. similar between the groups, underscoring the importance of meas- In this study, we have further developed the Matrigel assay to uring cytokines locally at the incipient tumour site (Supplementary quantify locally secreted cytokines during primary antitumour Fig. S2). immune responses. Using this method, we uncovered a common core of nine cytokines that were consistently associated with success- Cytokines associated with myeloma immunosurveillance. We ful cancer immunosurveillance.